Transient expression is widely used in functional genomics and molecular pharming because it avoids the long process of producing stable transgenic plants. Yet standard agroinfiltration is strongly biased toward Nicotiana benthamiana. In many crops and horticultural species, bacterial recognition activates salicylic acid (SA)-dependent immunity, while post-transcriptional gene silencing (PTGS) destroys messenger ribonucleic acid (mRNA) from introduced genes. Common reporters also create practical bottlenecks: green fluorescent protein requires external illumination, and luciferase assays typically need added substrates or destructive sampling. These limitations make cross-species optimization slow, expensive, and difficult to quantify over time. Given these challenges, deeper research is needed into broadly applicable, non-destructive systems that simultaneously improve gene delivery, transcript stability, and reporter sensitivity.
Led by Zhejiang University’s College of Agriculture and Biotechnology and ZJU-Hangzhou Global Scientific and Technological Innovation Center, with collaborators from several Chinese institutions and the Russian Academy of Sciences, the study was published (DOI: 10.1093/hr/uhag126) on 6 April 2026 in Horticulture Research. The researchers evaluated transformation-related factors across more than 20 plant families and developed an integrated platform combining the fungal bioluminescence pathway (FBP) reporter with NaP19, a module that couples the salicylate hydroxylase NahG with the viral gene-silencing suppressor P19 to enhance Agrobacterium-mediated transient expression across diverse vascular plants.
The team first examined whether plants could supply caffeic acid, the metabolic precursor required by the FBP. The broader survey included 42 species spanning algae and major land-plant lineages. Genomic and metabolite analyses showed that caffeic-acid biosynthesis is broadly conserved in land plants, although moss produced much weaker light than vascular plants and algae lacked the precursor. Using a charge-coupled device (CCD) camera, the researchers then screened immune suppressors and gene-silencing inhibitors without adding luciferin. NahG reduced salicylic acid and more than doubled expression, while P19 roughly doubled reporter output at an optimized optical density at 600 nanometers (OD600) of 0.25. When combined through a self-cleaving peptide, NaP19 produced about a tenfold increase in light in Nicotiana benthamiana, elevated transcripts of the FBP genes H3H and Luz, and reduced the cell damage caused by high P19 levels. Across nine representative crops and ornamentals, NaP19 raised luminescence by 1.39- to 14.21-fold. The platform also supported protein localization, luciferase complementation, and transcriptional assays in sunflower, mung bean, and three cotton genotypes, revealing native regulatory relationships that are difficult to test using stable transformation.
The authors said the platform was designed to remove several obstacles at once rather than improve only bacterial entry into plant tissue. By pairing a reporter that generates its own light with two effectors acting on different defense layers, they said researchers can see whether introduced genes are expressed and can test biological function in the same living tissue. The authors emphasized that this is especially useful for species where stable transformation is slow, genotype-dependent, or unavailable, because it creates a practical experimental window for examining proteins and regulatory networks in their native cellular setting.
The system could accelerate gene discovery in horticultural crops, support rapid validation of breeding targets, and broaden plant-based production of recombinant proteins and high-value metabolites. Because it works in native tissues, it may also help researchers test synthetic gene circuits, transcriptional regulators, and protein interactions before investing in stable transformation. Important limitations remain: fungal bioluminescence depends on endogenous caffeic acid, which can vary with species, stress, and metabolism, and the standardized infiltration conditions used here were not optimized for every plant. Future work should therefore add metabolism-independent internal controls across laboratories and growing seasons, tailor bacterial density and tissue conditions by species, and connect short-term expression changes with stable phenotypes, field performance, and scalable molecular-pharming workflows.
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References
DOI
10.1093/hr/uhag126
Original Source URL
https://doi.org/10.1093/hr/uhag126
Funding information
National Natural Science Foundation of China grants W2512082 and 32450506; Fundamental Research Funds for the Central Universities grant K20250113; Russian Science Foundation project 24-74-10087; the 78th General Program of the China Postdoctoral Science Foundation grant 2025M782776; and support from the Information Technology Center and State Key Laboratory of Computer-Aided Design and Computer Graphics at Zhejiang University.
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.